Neuropeptide Y (NPY) is known to be co-localized, with norepinephrine (NE) and adenosine triphosphate (ATP) in vascular sympathetic neurons where it is thought to play a role as a co-transmitter/co-modulator. Recent evidence has established that NPYplays a physiological role in sympathetically mediated vasoconstriction by acting on postjunctional Y1 receptors. NPY is also known to exert prejunctional effects leading to inhibition of NE and most likely ATP release and an increase in NPY-ir release via Y2 receptors as well as inhibition of NE synthesis via Y3 and Y5 receptors. These results suggest that the process of NE synthesis and release of sympathetic co-transmitters may be differentially modulated by NPY suggesting an additional level of control in the prejunctional regulation of sympathetic neurotransmission. However the physiological role of this action has not been completely established. We have obtained evidence that an important mechanism for the prejunctional effects of NPY is through inhibition of Ca 2+channels. There is also evidence for a role of SNARE proteins in the preferential release and modulation of transmitter releaseby auto and heteroreceptors. The purpose of the present proposal is to further investigate the physiological role (Aims 1 and 2) on the NPY induced modulation of sympathetic co-transmitter release and NE release and the mechanisms involved (Aim 3) in the prejunctional modulation by NPY and other mediators. The rationale for studies proposed in Aim 1 is as follows: If there is endogenous modulation of transmitter release by NPY, then agonists and antagonists specific for the prejunctional NPY autoreceptor should alter evoked transmitter release in a manner consistent with the receptors being activated by endogenous agonist (e.g. released NPY). In other words, the effect of exogenously administered agonists and antagonists should vary with the biophase concentration of endogenous NPY. The response of the effector cell should be consistent with inhibition or stimulation ofNPY release. It would also seem necessary to demonstrate functional receptors in vivo. A similar rationale exists for Aim 2. In Aim 1 we examine the effect of a series of selective Y1 and Y2 agonists and antagonists on the nerve stimulation evoked release of NE, NPY-ir and ATP as well as perfusion pressure in the mesenteric arterial bed. This will be done before and after the endogenous NPY concentration is elevated by: 1) increasing the frequency of nerve stimulation or 2) decreasing the perfusion rate; or 3) after the NPY concentration has been reduced by depletion of tissue levels acutely or chronically. We will also examine the in vivo effect of agonists and antagonists in the pithed rat preparation. In Aim 2 similar experiments will evaluate the effect of NPY drugs on the nerve stimulation evoked increase in NE synthesis as measured by DOPA accumulations after decarboxylase inhibition. In Aim 3 we will determine if interruption of SNARE proteins by Botulinum neurotoxins (BoNTs) inhibit the evoked release of NE, NPY-ir and ATP as a mechanism for preferential release or differentiated modulation. We will also examine if signaling through SNARE proteins contributes to the prejunctional modulation by NPY. These studies will provide useful new information on the functional role and mechanism of action of NPY in the prejunctional regulation of sympathetic neurotransmission.